Methods for inhibiting transcription of the cyclic AMP responsive element binding protein and the activating transcription factor 1

ABSTRACT

The present invention relates to members of the cyclic AMP responsive element binding protein/activating transcription factor 1 (CREB/ATF1) family of transcription factors. An embodiment of this invention constitutes an inhibitory agent (e.g., an antibody, small molecule or polypeptide) which binds to a fragment, spanning from about position 167 to about 181 of the amino acid sequence of the ATF1 protein bound to the target gene, with sufficient binding affinity to cause disassociation of ATF1 from the DNA of the target gene and/or prevent ATF1 from binding thereto through a currently unknown mechanism. As a result, transcription is prevented or, at least, inhibited, resulting in events of consequence to a virus or cell.

This is a divisional of application Ser. No. 08/210,880, filed Mar. 18,1994, now U.S. Pat. No. 5,641,486.

FIELD OF THE INVENTION

The present invention relates generally to members of the cyclic AMPresponsive element binding protein/activating transcription factor 1(CREB/ATF1) family of transcription factors. The consensus binding siteof these sequence specific transcription factors is present in manycellular and viral promoters. Because the CREB/ATF1 family of proteinsprovide unique targets for cancer and antiviral therapy, the inventionalso relates to methods for preventing transcription factor mediatedreplication of cancer cells or viruses. More particularly, the presentinvention relates to the inactivation or inhibition of thesetranscription factors through binding to newly identified, specificfunctional domains on the transcription factor that causes dissociationfrom the DNA to which it is bound, preventing gene transcription. Thepresent invention also relates generally to a method for inhibition ofCREB/ATF1 family mediated gene expression and to a method for preventingtranscription factor mediated viral replication or transcription factormediated aberrant cellular proliferation. The present invention alsorelates to a method for detecting molecules that inhibit the binding ofthe CREB family of transcription factors to DNA.

BACKGROUND OF THE INVENTION

Gene expression leading to the production of protein is most frequentlyregulated at the level of RNA production, and is termed transcription.Generally, control of transcription is mediated by activator orrepressor proteins termed transcription factors. A gene is transcribedafter a sequence of events determined by transcription factors hasresulted in positioning an enzyme (i.e., RNA polymerase) in the properlocation and configuration on the DNA. Transcription factors act throughat least two essential mechanisms: (i) binding to specific DNAsequences; or (ii) interacting with other proteins which subsequentlyinfluence transcription initiation. The DNA sequences which are involvedin regulation of either viral or eukaryotic gene expression and are thetargets for transcription factors occur in a variety of locations and atvarious distances from the transcriptional start and stop sites. TheseDNA sequences contributing to regulation consist of complex arrays ofrelatively short DNA sequence motifs. It is believed that tissuespecific gene expression occurs as a consequence of cooperation betweentranscription factors and the DNA sequences to which they bind. Eachmotif is a binding site for a specific family of transcription factors.

For the CREB family, the consensus binding site has been identified byMontminy et al., 1986 Proc. Natl. Acad. Sci., USA, 83: 6682-6686). Thissequence, TGACGTCA, is present in a wide variety of viral and cellulargenes, most notably ElA-inducible adenoviral genes and cAMP-induciblecellular genes. Some variation is found in the core sequence withretention of essential function. Specificity of CREB protein binding toparticular enhancers can be altered by interaction with viraloncoproteins, including Hepatitis B virus X (Maguire, H. F., et al,1991, Science 252:842-844), Human T-cell leukemia virus (HTLV-1) tax(Zhao, L. J., et al, 1992, Proc. Natl. Acad. Sci. USA. 89:7070-7074;Armstrong et al., 1993, Proc. Natl. Acad. Sci. USA. 90:7303-7307;Suzuki, et al., 1993, Proc. Natl. Acad. Sci. USA. 90:610-614.; Wagnerand Green, 1993, Science 262:395-399).

The CREB/ATF1 family of transcription factors is part of the bZIPsuperfamily of transcription factors. The most significant structuralsimilarity is the presence of a region with many basic amino acids (bregion), and a separate domain that allows close interaction with otherproteins with like structure, analogous to a zipper (ZIP). The basicdomain has a high concentration of the positively charged amino acidslysine and arginine, which form a tightly coiled alpha helix in thepresence of DNA. The basic domain lies in close proximity to a series ofamino acids in which leucine is present at every seventh position (theleucine zipper). Further, the leucine zipper forms an amphipathic alphahelix organized into coiled-coils with one surface being hydrophobic andthe opposite surface being hydrophilic. This provides for close pairingor dimerization with either identical proteins (homodimers) or similarproteins (heterodimers).

The numbering of the amino acid sequences for ATF1 used in thisapplication is based on the EuGene project designation of theATF1--Human (fragment) reported in Entry A34223. See also Hai T. et al,Genes Dev. 3:2083-2090 (1989).

SUMMARY OF THE INVENTION

The various embodiments of this invention hereinafter discussed arebased on the search for the existence of a functional domain on ATF1, amember of the CREB/ATF1 family which, when bound to a compoundrepresented by an ATF1 specific antibody, prevents ATF1 binding to DNA.This critical domain of ATF1 has been determined to be a peptidefragment, spanning from about position 167 to about position 181 of theamino acid sequence of the ATF1 protein. This fragment is locatedadjacent to the DNA binding region of ATF1, and is composed of thefollowing amino acid sequence: ##STR1##

Consequently, an embodiment of this invention constitutes an inhibitoryagent (e.g., an antibody, small molecule or polypeptide) which binds toa fragment, spanning from about position 167 to about 181 of the aminoacid sequence of the ATF1 protein bound to the target gene, withsufficient binding affinity to cause disassociation of ATF1 from the DNAof the target gene and/or prevent ATF1 from binding thereto through acurrently unknown mechanism. As a result, transcription is prevented or,at least, inhibited, resulting in events of consequence to a virus orcell. It is preferred that the inhibitory agent target a region of saidfragment having no more than about 8 amino acids because a smallercompound is more stable, is more capable of entering cells, and hasreduced side effects.

The inhibitory compounds of this invention are further characterized bytheir ability to inhibit the shift or the supershift of DNA/ATF1 in theATF Inhibition Detection Assay (AIDA) of this invention, as hereinaftermore fully described. This assay provides a convenient method for thedetection of compounds that bind to the target gene with sufficientbinding affinity to cause disassociation of ATF1 from the DNA of thetarget gene.

Another aspect of the present invention relates to a method forpreventing, ex vivo or in vivo, transcription factor mediatedreplication of cancer cells or viruses, comprising exposing said cellsor viruses, ex vivo or in vivo, to an effective amount of an inhibitoryagent of this invention. Said agent binds to a portion of thetranscription factor, such as the region in ATF1 spanning from aboutposition 167 to about 181 with sufficient binding affinity to causedisassociation of the transcription factor from the DNA of the targetgene and thereby prevent transcription. The inhibiting compound can bean antibody, subcomponents of the antibody (e.g., Fab fragments or sFvsubunits), a polypeptide representing the configuration of the antibodybinding site, or small molecules that also resemble the configuration ofthe antibody binding site; provided that in each case the inhibitingcompound is capable of binding specifically to the intended criticaldomain on the transcription factor (e.g., the critical domain of ATF1previously discussed) with consequent prevention or inhibition oftranscription.

Another related embodiment is a method for preventing other CREB/ATF1proteins from binding to cellular DNA, comprising exposure of said DNAto an effective amount of a compound which binds to a portion of theCREB/ATF1 proteins and prevents binding of the CREB/ATF1 proteins toDNA, preventing transcription. Especially preferred is a method fordisassociating CREB/ATF1 proteins from DNA comprising exposing said DNAto an effective amount of a compound which specifically binds to aportion of mammalian CREB proteins and results in disassociation. Forthe CREB/ATF1 protein, ATF1, the binding domain has been determined tobe SEQ ID NO: 1, representing a domain within 5 amino acids of the alphahelix DNA binding region of CREB family protiens.

Thus, a related embodiment of this invention is a method for inhibitingCREB/ATF1 family protein mediated transcription comprising exposure ofthe CREB/ATF1 protein bound DNA to an effective amount of a compound ofthis invention. The inhibitory compounds of this invention arecharacterized by their ability to supershift DNA/ATF1 in the ATFInhibition Detection Assay (AIDA) of this invention, as hereinafter morefully described.

In one embodiment of this invention and, as is demonstrated hereinafter,the inhibitory compound of this invention can be a monoclonal antibodyor a subcomponent of a monoclonal antibody. Exemplary of suchsubcomponents are Fab fragments or sFv subunits of the monoclonalantibody. The Fv element is thought to be the smallest component of anantibody that is capable of binding to the original epitope and derivedsFv proteins have been shown to have binding affinities equivalent tothe parent monoclonal antibody (Bird, et al, Science 242: 423-426,1988).

Although inhibition using a monoclonal antibody (MAb), herein referredto as MAb 4, is hereinafter demonstrated, because of its size a MAb isnot an ideal inhibitory therapeutic agent. Consequently, it is preferredto use the above discussed small fragments or subunits of the MAb or,alternatively, to employ a small peptide or other small molecule whichbinds to the ATF1 epitope depicted by Sequence No. 1. Such peptides canbe detected conveniently using the AIDA assay of this invention, as ismore fully described hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C and 1D. Shows the results of MAb 1 and MAbs 3-5 inimmunoblot assays as described in Example 1. (The preparation of theseMAbs is described hereinafter.)

FIGS. 2A, 2B, 2C and 2D. Shows the results of the DNA binding assay withthe MAb 1 and MAbs 3-5 panel and IgA and IgG1 antibodies as described inExample 3.

FIG. 3. Shows the promoter templates for the in-vitro PCNA transcriptionstudies described in Example 4.

FIG. 4. Shows the effects of the MAb on in-vitro PCNA transcription asdescribed in Example 4.

FIG. 5. Shows the regions of interest on CREB and ATF1.

FIG. 6. Shows MAb 1 and MAbs 3-5 reactivity with major thrombinfragments of recombinant ATF1 as described hereinafter in Example 5.

FIG. 7. Shows the DNA binding analysis with thrombin-digested ATF1:undigested ATF1 (lane 1), digested ATF1 (lane 2), digested ATF1 with 30×unlabeled CRE competitor (lane 3) or MAb 1 and MAbs 3-5 (i.e., M1 andM3-5, lanes 4-7), as described in Example 5.

FIG. 8. Shows a graph of peptide c binding of MAb 4 by competitiveinhibition ELISA as described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

Examples 1-7 demonstrate the method for detecting ATF1-inhibitorycompounds and inhibiting in-vitro transcription. The examples alsodemonstrate the use of an ATF Inhibitory Detection Assay (AIDA), ashereinafter described, to select an inhibitory compound of thisinvention and use of that compound to prevent transcription factormediated replication of cancer cells.

The following preparations and methodologies are those utilized inexamples 1-8.

Preparation of Recombinant CREB

Recombinant CREB was produced using CREB coding sequences, preparedaccording to L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA,89:7070-7074, 1992. The cDNA for CREB was cloned according to themethodology of Studier et al, Methods Enzymol., 185:60-89, 1990, at theNdeI/BamHI sites of the pET-11a expression plasmid. The protein wasexpressed from the bacteriophage T7 promoter and was purified fromEscheria coli cell lysates on DNA-cellulose columns (Sigma, Catalog#D8515).

Preparation of Recombinant ATF1

Recombinant ATF1 was produced using expression vectors containing fulllength ATF1, according to L. J. Zhao and C. Z. Giam, Proc. Natl. Acad.Sci. USA, 89:7070-7074, 1992. The cDNA for ATF1 was cloned according tothe methodology of Studier et al, Methods Enzymol., 185:60-89, 1990, atthe NcoI/BamHI sites of pET 11d. The protein was expressed from thebacteriophage T7 promoter and was purified from Escheria coli celllysates on DNA-cellulose columns (Sigma , Catalog #D8515).

Preparation of Nuclear Extracts

Nuclear extracts were prepared from 1-5×10⁸ cells as described by Dignamet al., Nucleic Acids Res. 11:1475-1489, 1983, and dialyzed against 20mM HEPES (N- 2-Hydroxyethyl!piperazine-N'- 2-ethanesulfonic acid!), pH7.9, 100 mM KCl, 2 mM dithiothreitol, 20% glycerol, 0.2 mM EDTA(ethylenediamine tetraacetic acid), 1 mM PMSF (phenylmethylsulfonylfluoride), 20 μg/ml aprotinin and 10 μg/ml trypsin-chymotrypsininhibitor. Alkaline phosphatase treated nuclear proteins were preparedby digesting nuclear extracts (150 μg protein/reaction) with 20 units ofcalf intestine alkaline phosphatase (New England Biolabs, Catalog #2905,1993) in 50 mM Tris, pH 9.5, 50 mM NaCl, 5 mM MgCl₂ at 37° C. for 1 hr.Total protein was determined by the Bradford Assay (Biorad, Catalog#500-0002, 1993) and amounts of ATF1 and CREB were estimated by Westernblot analysis, as hereinafter described.

Preparation of monoclonal antibodies

The ATF1 monoclonal antibodies were generated using three 10 μginjections of recombinant ATF1, prepared as described above, asimmunogen with the Ribi Adjuvant System (Ribi Immunochem Research Inc.,Product Code #730, Masihi, K N et.al. Int. J. Immuno Pharmacol.,8:339-345, 1989). The method used for generating the monoclonalantibodies was that of Kohler, G. and Milstein, C. (Eur. J. Immunol.6:511-519, 1976) The panel of MAbs were screened initially by ELISA(Volker and Bidwell, Manual of Clinical Laboratory Immunology, 3rdEdition, Chapter 17, 1986) on plate-bound recombinant ATF1. Isotypeswere determined using a kit from Amersham (Product #RPN-29). All MAb hadκ light chains, MAb 1, 3 and 4 were IgG1 isotype and MAb 5 was an IgAisotype. Antibody affinity was evaluated by competitive ELISA (Friguetet al., J. Immunol. Methods, 77:605-319, 1985) using recombinant ATF1 asan antigen.

IgG1 MAb used in DNA binding and in vitro transcription assays wereaffinity purified on a protein G column and quantitated by spectroscopyat A₂₈₀ and the Bradford Assay (Biorad protein assay, Catalog#500-0002). IgA antibodies in ascites fluid were quantitated by scanningIgA light chain on dried coomassie blue stained SDS-PAGE gels with aScanMaker 600ZS (Microtek, Inc.) and analyzed using the "Image" programon a Macintosh IIci computer. This analysis determined that the MAbs IgAconcentration was 10 mg/ml whereas the control was 15 mg/ml.

Anti-CREB antibody used for western blot analysis and DNA binding assayswas a rabbit polyclonal antibody against the CREB α-peptide (Santa CruzBiotechnology, Catalog #SC-58). Isotype matched myeloma proteins IgG1, κ(MOPC 15) (SIGMA), and IgA, κ (TEPC 15) were used as negative controlsfor the MAb assays.

Western Blot Analysis

Proteins were resolved by SDS-PAGE electrophoresis on 15% polyacrylamidegels and transferred to nitrocellulose. Nonspecific binding was blockedwith 10% powdered milk in Tris buffered saline plus 0.1% TWEEN 20 andmembranes were incubated for 1 hour with hybridoma tissue culturesupernatants. Supernatant from each of the monoclonal cultures 1,3, 4,and 5 were used separately and prepared in accordance with themethodologies set forth above, diluted 1:4 in Tris buffered saline.Bound antibody was detected with a commercialbiotin-streptavidin-enhanced detection kit (Amersham, Product #RPN-22,1993) used according to the manufacturer's instructions.

EXAMPLE 1

The following example demonstrates that MAb 1, 3, 4, and 5 react withuntreated or alkaline phosphatase treated ATF1 on Western immunoblots ofnuclear extracts from human and murine cell lines.

Immunoblotting

The MAb were tested as reagents for immunoblotting. Nuclear extracts (15μg per lane) from HeLa human cervical epithelioid carcinoma cells (H),L929 murine connective tissue fibroblasts (L), or MT-4 HTLV-1transformed human T cells (M) were analyzed on 15% SDS-PAGE gels with(+) or without (-) calf intestine alkaline phosphatase (Alk Phos)treatment. rC indicates purified recombinant CREB protein (15 ng perlane).

Results indicate that all 4 MAb react with untreated or alkalinephosphatase treated ATF1 on Western immunoblots of nuclear extracts fromhuman and murine cell lines (FIGS. 1A, 1B, 1C and 1D). ATF1 also wasreadily detected in whole cell extracts from established cell lines.Only MAb 1 reacted with phosphorylated and dephosphorylated CREB innuclear extracts. MAb 1, 3 and 5 detected as little as 0.5-1 ng ofrecombinant ATF1 on immunoblots; however 5-10 ng was required forreaction with MAb 4.

ATF1 INHIBITION ASSAY ATF Inhibition Detection Assay (AIDA)

Whether an antibody or compound constitiues an inhibitory agent of thisinvention can be determined by testing the antibody or compound in theAIDA. This assay evaluates the candidate agent for its ability toinhibit ATF1 binding to DNA in the electrophoretic mobility shift assayherein referred to as the AIDA. The AIDA is generally simpler, faster,and more sensitive than other methods for detecting sequence-specificDNA-protein binding. Separate lanes of the gel are used for thefollowing compounds respectively: 1) DNA alone; 2) DNA with ATF1; 3) DNAwith ATF1 and the agent to be tested. The gels are runelectrophoretically to determine which compounds result in disruption ofa shift or supershift of the DNA. Larger molecules shift to a higherposition on the gel and each complex produces a different and uniquepattern. The use of the AIDA to identify an inhibitory agent of thisinvention, as exemplified by MAb4, is shown in Example 2.

EXAMPLE 2 DNA BINDING

This example demonstrates that binding of MAb4 to the novel domain(epitope) of this invention inhibits ATF1-DNA binding.

DNA Binding Assays

The double-stranded oligonucleotides used in the electrophoreticmobility shift assays, obtained from Promega were as follows: CRE:5'-AGAGATTGCC TGACGTCA GAGAGCTAG-3' (SEQ ID #2) (CRE Catalog #E3281),AP1: 5'-CGCTTGA TGAGTCA GCCGGAA-3' (SEQ ID #3) (AP1 Catalog #E3201). DNAbinding mixtures (20 μl containing 10-20 ng recombinant ATF1 and/orCREB, 1 μg poly dI-dC!, and 2.5 μg bovine serum albumin in 10 mM Tris,pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 1 mM MgCl₂, 4% (by volume)glycerol, and 0.035 picomoles ³² P-labeled probe) were incubated for 20min at room temperature, then run on native 4% polyacrylamide gels inhigh ionic strength buffer (25 mM Tris, 190 mM glycine, 1 mM EDTA) at 4°C.

DNA binding assays with recombinant ATF1 and CREB (FIGS. 2A, 2B, 2C AND2D) demonstrated that MAb 1 supershifts both ATF1 and CREB complexes tothe same extent, and MAb 3 shifts CREB a lesser distance than ATF1. MAb4 prevented ATF1-DNA binding, even if it was added after the DNA probe,but supershifted CREB. MAb 5 supershifted ATF1 and did not react withrecombinant CREB.

Decreasing amounts of each MAb were used in the DNA binding assay todetermine ATF1 affinity. MAb 1 has the highest affinity in this assay,with 0.020 μg of MAb (0.5:1 molar ratio of divalent antibody molecule toATF1 monomer) completely supershifting 0.010 μg of ATF1. Two μg of MAb 3(50:1 molar ratio) or 5 μg of MAb 5 (100:1 molar ratio) supershiftedATF1 to the slower migrating band (Supershift II) and 0.5 μg of MAb 4(12:1 molar ratio) completely prevented 0.010 μg of ATF1 from binding tothe probe. Limiting amounts of MAb 3 or 5 with ATF1 produced a fastermigrating shifted band (Supershift I) at the same mobility as the MAb 1ATF1/CREB supershift or the MAb 3 or 4 CREB supershift. Shifting all ofthe ATF1 to at least this level required 0.05 μg of MAb 3 (1:1 molarratio) or 0.20 μg of MAb 5 (4:1 molar ratio).

Although not wishing to be bound by theory, it is believed thatSupershift I represents one antibody molecule bound to eachtranscription factor dimer and Supershift II represents two moleculesbound to each transcription factor dimer. A tenfold higher concentrationof MAb 1 and fifty-fold higher concentrations of MAb 3 and 4 wererequired for CREB supershifts as compared to ATF1 supershifts orATF1-DNA complex blocking. Reaction of MAb 3 and 4 with CREB in the DNAbinding assay was surprising because these antibodies did not react withCREB on dot blots, even if CREB was pre-incubated with unlabeled CREoligonucleotide.

Results of preliminary DNA binding experiments with HeLa cell extractsdemonstrated that MAb 1, 3 and 5 supershifted most of the CRE bindingprotein. MAb 3 or 5 (5-10 μg) produced two shifted complexes and a smallamount of unshifted complex remained in reaction mixtures containing MAb5. Because of the high level of ATF1 produced, most of the CREB in HeLanuclear extracts exists as ATF1-CREB heterodimers (Hurst et al., NucleicAcids Res., 19:4601-4609, 1991). Again not wishing to be bound bytheory, it is believed the MAb 5 supershifted complexes represent ATF1homodimers and ATF1-CREB heterodimers, and the unshifted materialrepresents CREB-CREB homodimers. MAb 4 reduced the total amount ofshifted complexes, indicating that it prevents cellular ATF1 binding andmay shift or prevent heterodimer binding, depending on the relativeamount of antibody and ATF1 and CREB homo- and heterodimers in thesolution.

EXAMPLE 4 IN-VITRO TRANSCRIPTION PCNA In-Vitro Transcription

Effects of MAb on transcription were evaluated using the HeLa nuclearextract in vitro transcription system from Promega according to themanufacturer's instructions (Promega, Catalog #E3110, Protocol #TB123,1992) except that amounts of MgCl₂ (5 mM) and rATP (0.30 mM) wereoptimized as described by P. J. Farnham and R. T. Schimke (Mol. Cell.Biol., 6:2392-2401, 1986) and reactions were incubated at 26° C. for 1hr. Antibody was incubated with nuclear extract and MgCl₂, for 30 minbefore adding rNTP's and template. Promoter templates (FIG. 3) wereProliferating Cell Nuclear Antigen (PCNA) luciferase expression vectorconstructs. PCNA 5 contains -182 to +143 of the PCNA promoter, includingCRE/CRE, TGGCGTCA/TGACCTCG, (SEQ ID #5). PCNA 2 is a truncated constructcontaining only the CRE/CRE and PEA3 sites (-80 to +143) and mP-5 is aPCNA-5 construct with both CRE elements mutated to AGGGGTCA/AGAGCTCA(SEQ ID #6).

Specific PCNA RNA transcription was detected using an ³² P-labeledprimer (5'-GACTAGATGAGAGCTACTCTAAGAGGAACG-3') (SEQ ID #4) (EMBL DataLibrary, Accession=X53068) antisense to +97 to +127 of the PCNA gene(Shipman-Appasamy et al., DNA Seq., 2:181-191, 1991), prepared inaccordance with the methodologies of Beaucage and Caruthers, TetrahedronLetters, 22:1859-1862, 1981: and Sinha et.al., Tetrahedron Letters,24:297-300, 1983). RNA transcripts were annealed with the primer in 10mM Tris-HCl, pH 8, 1 mM EDTA, at 70°-75° C. for 10 minutes and cooled toroom temperature for 10 minutes.

Reverse transcriptase buffer provided by the manufacturer was added andthe solution was adjusted to 0.01 mM dithiothreitol, and 0.5 mM each ofdATP, dTTP, dGTP, dCTP. Each 30 μl reaction was warmed to 42° C., 1 μlcontaining 200 units of SUPERSCRIPT™ RNase H⁻ reverse transcriptase(BRL, Catalog #18053-017) was added, and incubated for 30 minutes.Denaturing gel buffer, 20 μl, (98% formamide, 10 mM EDTA, 0.1% eachxylene cyanol and bromophenyl blue) was added, samples were heated to90° C. for 10 minutes and analyzed by electrophoresis on 6% acrylamidegels containing 7M urea in 90 mM Tris-borate, 1 mM EDTA.

The labeled 127 bp product was sized by comparison with ΦX174 Hinf Imolecular weight markers from Promega (Catalog #E3511) and quantitatedon dried gels with a Betascope 603 Blot Analyzer (Betagen Corp.,Walthan, Mass., 1989) according to the manufacturer's instructions.

The effects of the panel of MAb on in vitro transcription using themurine proliferating cell nuclear antigen (PCNA) gene promoter astemplate were evaluated. The PCNA protein is expressed at much higherlevels in proliferating cells than in quiescent cells, and is aco-factor for DNA polymerase delta, functioning in DNA replicationduring S phase. PCNA RNA transcription increases in interleukin-2 (IL-2)stimulated T-cells during G1 phase progression, but PCNA MRNA levels areregulated by changes in MRNA stability in serum stimulated murine 3T3fibroblasts (Shipman-Appasamy et al., DNA Seq., 2:181-191, 1991).

When added to HeLa cell nuclear extracts in the PCNA in vitrotranscription system, MAb 4 reduced transcription to 5% of reactionswith no added antibody, MAb 1 increased transcription 1.5 fold and MAb3, 5 or control antibodies did not significantly affect transcription(FIG. 4). In preliminary experiments with murine cell nuclear extracts,MAb 4 also inhibited transcription. Transcription was reduced to 6% witha template containing mutated CRE elements, and was not detectable witha truncated template containing only CRE and PEA3 elements. Addition ofMAb 4 at a approximately the same molar ratio as that required toprevent ATF1-DNA binding (12:1 molar ratio of divalent MAb to monomericATF1) reduced specific in vitro transcription to the same extent asmutating the CRE elements.

EXAMPLE 5 EPITOPE MAPPING Mapping of MAb Epitope

Because each MAb produces a different pattern in the DNA binding assayand two MAb (#1 and #4) have opposite effects on in vitro transcription,the location of the MAb epitopes within the ATF1 molecule wasdetermined. The first step in determining the fine specificity of theMAb was to cleave recombinant ATF1 into large fragments.

Testing several enzymatic and chemical cleavage methods determined thatthe best results were obtained with thrombin digestion. Two majorcleavage products, with apparent molecular weights on SDS-PAGE of 22 kDand 14 kD, were produced.

For MAb epitope mapping, >95% pure (by SDS-PAGE) recombinant ATF1,purified on a DNA-cellulose column, was digested for 40 or 80 hours withhuman thrombin (3806 NIH units/mg, Calbiochem catalog #605195) in 50 mMTris pH 8.0, 5 mM EDTA, 1 mM dithiothreitol at 37° C, adding 0.4-1 unitof thrombin at 8-24 hour intervals. Digests were analyzed by SDS-PAGEand Western immunoblotting and major proteolytic fragments wereidentified by protein sequencing from electroblots as described byMatsudaira, P. (J. Biol. Chem. 262:10035-10038, 1987). The eight aminoterminal amino acids of each fragment were determined and compared withthe known ATF1 sequence. The 22 kD fragment contained the amino terminusof ATF1 described by Yoshimura, T., et.al. (EMBO J. 9:2537-2542, 1990)and Rehfuss, R. P., et.al. (J. Biol. Chem/266:18431-18434, 1991). Theamino terminal sequence of the 14 kD fragment indicated that it was thecarboxy terminal portion of ATF1 and that the major thrombin digestionsite is after arginine 144 in the partial sequence described by Hai, T.et. al. (Genes and Dev. 3:2083-2090, 1989).

Immunoblotting and DNA binding analysis of thrombin-digested ATF1indicated that MAb 1 and 3 react with the amino-terminal half of themolecule which contains domains involved in transcriptional activation(FIG. 5) and MAb 4 and 5 reacted with the carboxy-terminal half whichincludes the leucine zipper and DNA binding region.

MAb 1, 4, and 5 also react with a less abundant 29 kD fragment whichdoes not react with MAb 3 (FIG. 6). This 29 kD fragment may be producedwhen ATF1 is digested at a consensus thrombin site within the P-box,removing 78 amino terminal amino acids. Reaction of this fragment withMAb 1 but not MAb 3 indicates that MAb 3 reacts with the amino terminalregion and MAb 1, reacts with a centrally localized epitope on ATF1.

Identity of the major fragments was confirmed by DNA binding analysis(FIG. 7). MAb 1 and 3 did not affect fragment-DNA bindng, MAb 4prevented binding, and MAb 5 supershifted bound fragments. Concentratingon the shorter 14 kD DNA binding fragment, overlapping syntheticpeptides were produced, representing the areas within this fragment thatdiverge between ATF1 and CREB.

EXAMPLE 6 PEPTIDE c REACTIVITY MAb4 and MAb5 Reactivity with Peptide c

MAb 4 and 5 reactivity was analyzed by dot immunoblotting andcompetitive ELISA. Focusing on the shorter 14 kD DNA binding fragmentwhich reacts with MAb 4 and 5, overlapping synthetic peptidesrepresenting the areas within this fragment that diverge between ATF1and CREB were produced (FIG. 5). Peptides were synthesized via Fmocprocedures on a p-hydroxymethylphenoxymethyl polystyrene (HMP) resinsupport. After synthesis and oxidation the peptides were deprotected andcleaved from the resin by standard acidolysis in trifluoracetic acid andpurified by reverse-phase HPLC methods. In FIG. 8 peptides represent thefollowing ATF1 amino acids: a(▴) : 145-159 TTPSATSLPQTVVMT (SEQ ID #7);b(∘): 156-170 VVMTSPVTLTSQTTK (SEQ ID #8); c(): 167-181 QTTKTDDPQLKREIR(SEQ ID #9); d(♦): 147-166 PSATSLPQTVVMTSPVTLTS (SEQ ID #10); and e(58): 219-233 EELKTLKDLYSNKSV (SEQ ID #11). MAb 4 and 5 reactivity wasanalyzed by dot immunoblotting and competitive ELISA. On the dot blots,MAb 4 reacted strongly with peptide c and MAb 5 reacted weakly withpeptide d.

In the competitive ELISA, peptide c inhibited MAb 4 binding to ATF1 evenmore efficiently than the intact ATF1 protein (▪ FIG. 8). The otherpeptides did not affect MAb 4 binding. None of the peptides inhibitedMAb 5 binding to ATF1 in ELISA. These assays identified the MAb 4epitope within the 10 amino acids amino proximal to the DNA bindingregion (amino acids 205-219, peptide c). However, although the MAb 5epitope may be within peptide d, it is not accurately represented by thesynthetic peptide and may be similar to a discontinuous epitopedescribed by Szilvay, A. M., et.al. (Arch. Virol. 131:393-403, 1993).

From the foregoing discussion and experimental results, it can be seenthat another embodiment of the subject invention is a method fortreating an individual having an ATF1-mediated disease comprisingadministering to said individual an effective amount of a compositionselected from the group consisting of an antibody and a peptide; saidcomposition capable of specific binding to SEQ ID NO:1 and regulatingtranscription, and further characterized by exhibiting a therapeuticallyuseful change in ATF1-mediated cell behavior. The inhibitory agents ofthis invention are administered to said individual (e.g., a human orother mammalian host) in an amount and dosage form effective to inhibitand/or block ATF1 activity. The structures of the inhibitory agents arebased on the sequence and structure of the protein target and thecomplementarity determining region (CDR) of the antibody whichrecognizes a specific domain (epitope) near the DNA binding region ofthe ATF1 alpha helix domain and blocks transcription. For example, asdemonstrated above, the MAb 4 antibody blocks the formation ofDNA-protein complexes and interferes with in-vitro transcription.Likewise, the inhibitory agents of the invention bind to the epitopicsite on ATF1 and block ATF1-mediated activity. In one embodiment theinhibitory agent is composed of a peptide fragment selected or designedto bind to the essential site on ATF1.

For in vivo use the inhibitory agents of this invention are preferablyadministered in pharmaceutically acceptable dosage forms which containin unit dosage form an effective amount of the inhibitory agent. In suchpharmaceutically effective unit dosage forms, the inhibitory agents ofthis invention exhibit excellent and effective therapeutic activity inthe treatment of ATF1-mediated diseases. Exemplary is the treatment ofHTLV1 mediated leukemic cell proliferation. Obviously, the actual dosesemployed can vary and in general depend on the particular disease beingtreated.

A decided practical advantage is that the active inhibitory compound canbe administered in a convenient manner, such as by the oral, intravenous(where water soluble), intramuscular, subcutaneous, intranasal,intradermal or suppository routes. Depending on the route ofadministration, the active ingredients which comprise an ATF1 inhibitoryagent of this invention may be required to be coated in a material toprotect said ingredients from the action of enzymes, acids and othernatural conditions which may inactivate said ingredients.

In a preferred embodiment, the therapeutically effective inhibitoryagent is a cyclic polypeptide containing the amino acid sequence of thecomplementarity determining region (CDR) of MAb 4. The CDR-containingpeptide can be produced using standard techniques for isolating cDNA andexpressing the peptide in standard systems and techniques from theliterature. The full monoclonal antibody recognizes SEQ ID NO: 1 and theCDR derived therefrom and reconstructed as a single chain recombinantpolypeptide also recognizes SEQ ID NO: 1. The length of the polypeptidecan be varied, provided that such variation does not negate its abilityto recognize SEQ ID NO: 1 or otherwise interfere with its pharmaceuticaleffectiveness. In general, it is not practical for the length of thepolypeptide to exceed 20 amino acid residues.

To increase the activity and solubility of the recombinant construct,the side chains of the polypeptide can be modified or pharmaceuticallyacceptable salts can be employed. Such side chains generally can vary inlength from between about 3 to about 8 carbons.

This preferred polypeptide can be synthesized for use as either an oralsolution or an intravenous solution. The preferred mode ofadministration is orally. Blood concentrations will vary but generallywill peak between 2 and 4 hours after administration. The absolute levelwill be determined by the dose which is expected to range between 500and 1400 mg/ml. The dosage is individualized based on projected bloodvolume or body weight of the patient. In general, a dose of betweenabout 10 and about 20 mg/kg is effective, although higher or lower dosescan be employed. When used as an antineoplastic preparation, dailydosing continues until a therapeutic regression of the tumor is seen. Incases of utilization of the therapeutic compound for treatment ofinflammatory or infectious conditions, a lower dose, generally on theorder of about 5 mg/kg, can generally be employed.

STATEMENT OF DEPOSIT

A deposit of the hybridoma cell line C41-4.1B disclosed above has beenmade with the American Type Culture Collection (ATCC), 12301 ParklawnDrive, Rockville, Md., 20852. The date of deposit was Oct. 24, 1996. Allrestrictions upon the deposit have been removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. §1.801-1.809. TheATCC accession number is ATCC HB-12218 as evidenced by the attachedreceipt from ATCC. The deposit will be maintained in the depository fora period of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replacedas necessary during that period.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlnThrThrLysThrAspAspProGlnLeuLysArgGluIleArg                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AGAGATTGCCTGACGTCAGAGAGCTAG27                                                 (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CGCTTGATGAGTCAGCCGGAA21                                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GACTAGATGAGAGCTACTCTAAGAGGAACG30                                              (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AGGGGTCAAGAGCTCA16                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TGGCGTCATGACCTCG16                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ThrThrProSerAlaThrSerLeuProGlnThrValValMetThr                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ValValMetThrSerProValThrLeuThrSerGlnThrThrLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlnThrThrLysThrAspAspProGlnLeuLysArgGluIleArg                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ProSerAlaThrSerLeuProGlnThrValValMetThrSerProVal                              151015                                                                        ThrLeuThrSer                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GluGluLeuLysThrLeuLysAspLeuTyrSerAsnLysSerVal                                 151015                                                                        __________________________________________________________________________

I claim:
 1. An inhibitory compound which binds to a fragment spanningfrom about position 167 to about position 181 of the amino acid sequenceof the ATF1 protein with sufficient binding affinity to causedisassociation of ATF1 from the DNA of the gene to which ATF1 is boundand prevent transcription.
 2. Composition of claim 1 wherein saidfragment is the ATF1 epitope having the following sequence: ##STR2## 3.The inhibitory agent as in any one of claims 1-2 wherein said inhibitoryagent is an antibody.
 4. Inhibitory agent of claim 3 wherein saidantibody is a monoclonal antibody.
 5. An antibody, or antibody fragment,specific for SEQ ID NO:1.
 6. The monoclonal antibody of claim 4,specific for SEQ ID NO:1.
 7. The inhibitory agent as in any one ofclaims 1-2 wherein said inhibitory agent is a peptide.
 8. Peptide ofclaim 7 wherein said peptide has between about 8 and about 20 aminoacids.
 9. A peptide fragment which contains a sequence comprising theamino acid sequence spanning from about position 167 to about 181 of theATF1 protein, said fragment having between about 8 and about 30 aminoacid moieties, and binding specifically with the antibody of claim 5.10. The fragment of claim 9 having no more than 8 amino acid moietiescorresponding to SEQ ID NO:1.